The processing of antigens by antigen-presenting cells (APCs) takes place by two different routes. Antigens occurring inside the cell are presented by MHC I (major histocompatibility complex class I, MHC class I) molecules on the cell surface, whereas extracellular antigens are presented by MHC II (major histocompatibility complex class II, MHC class II) molecules on the cell surface. Both mechanisms initiate an immune response by the host to the antigen. The route taken by the antigen from uptake into the cell until presentation on the cell surface in the form of an MHC II-antigen complex proceeds via various cell organelles, inter alia via the endoplasmic reticulum, the Golgi apparatus, the trans-Golgi network, lysosomes, endosomes and via MHC class II compartments (MIICs). The MIICs play an important part in the MHC II-mediated antigen presentation. In these organelles of the cell, the MHC II molecules are loaded with low molecular weight antigens or with proteolytic fragments of proteins. In this process, the invariant chain (also called MHC II gamma chain or Ii) which is initially bound to the MHC II molecule undergoes proteolytic degradation, and the antigen is bound to the MHC II molecule under the regulation of various proteins which bind directly or indirectly to MHC II. These regulatory molecules in humans include, inter alia HLA-DM, HLA-DO, LAMP-1, LAMP-2, CD63, CD83, etc. The exact function of these proteins is in part unexplained as of yet, but many of them have signal sequences which promote their transport to the lysosomes, to the endosomes, to the trans-Golgi network, to the MIICs, etc. A number of proteases are involved in the proteolytic processing necessary, so that the antigen can be presented on MHC II molecules. The proteases present in MIICs include, inter alia various members of the cathepsin family such as, for example, cathepsin S and cathepsin L. In species other than humans, the homologue MHC Class II molecules have different amino acid sequences. In Equine animals the organization of the MHC system is similar to humans with contiguous class I, III, and II regions (Kelley J et al., Immunogenetics 2005, 56: 683-695).
The concept of providing modular antigen transportation (MAT) molecules for modulating immune responses, associated constructs, method and uses thereof is disclosed in WO 2004/035793 (US equivalent US 2005/0281816), hereby incorporated by reference. This document describes the usefulness of a three-part-molecule, the MAT molecule, for introducing epitopes of antigens into cells, thus, determining the immune response to be modulated by such MAT molecule. Therein, various translocation modules, targeting modules as well as antigen modules are described. This technology and its underlying method make it possible, firstly, to convey antigens efficiently from the extracellular space into the intracellular space of a target cell, and, secondly, make it possible for the antigens, after arrival in the interior of the cell, to reach efficiently cell organelles in order to be subsequently processed for antigen presentation. Generally, the two-stage process can be utilized for the targeted, efficient modulation of the immune response in a subject. The use of MAT molecules is disclosed for example in Martínez-Gómez J M et al. [Allergy 2009, 64(1): 172-178]; Rose H (Arb Paul Ehrlich Inst Bundesinstitut Impfstoffe Biomed Arzneim Langen Hess, 2009, 96, 319-327) as well as recently in Senti G et al. [J Allergy Clin Immunol., 2012, 129(5): 1290-1296]. Based on the MAT technology, the major cat allergen Fel d1 was fused to a TAT-derived protein translocation domain and to a truncated invariant human chain for targeting the MHC class II pathway. Immunogenicity was evaluated in mice, while potential safety issues were assessed by suitable tests based on basophil reactivities from cat-dander-allergic patients. The possible use of this model compound has been demonstrated. It is described therein, that it is expected that MAT molecules are safer and more efficient in inducing the desired immune response, namely hyposensitization, than recombinant allergens or allergen extracts in conventional allergen-specific immunotherapy (SIT). In the recent publication by Senti G. et al. intralymphatic immunotherapy for cat dander allergy in humans inducing tolerance after three injections was described. Therein, a first-in-human clinical study with the MAT-Fel d1 was described, demonstrating safety and induction of allergen tolerance after intra-lymphatic injection of three injections only.
Further prior art is as follows:
Gadermaier G et al. (Molecular Immunology 2010, 47: 1292-1298) describe the targeting of the cysteine-stabilized fold of Art v1 for immunotherapy of Artemisia pollen allergy. The authors used genetic engineering approaches for targeting Art v1 posttranslational modifications aiming at the creation of hypoallergenic molecules: (i) disulfide bridges of the defensin domain were disrupted by site-directed mutagenesis and (ii) the mutant constructs expressed in E. coli for the production of non-glycosylated proteins. However, the objective was clearly only manipulating the three-dimensional fold of the Art v1 defensin domain to abrogate IgE-binding (i.e. creating a hypoallergenic molecule) by exchanging single cysteine residues for serine—while keeping intact (i.e. unmodified) the recognized T-cell epitopes (even if such contain cysteine residues).
The report of the 3rd Havemeyer workshop on allergenic diseases of the Horse (Hólar, Iceland, June 2007, Veterinary Immunology and Immunotherapy 2008, 126: 351-361) focused on immunological and genetic aspects of insects bite hypersensitivity (IBH) and recurrent airway obstruction (RAO). At this workshop, novel approaches for SIT against IBH were discussed, among others, the use of viral vectors or protein vaccination with allergens coupled to modular antigen translocating (MAT) molecules.
In SIAF Annual Reports 2010 and 2011 Crameri R reports the use of MAT technology for vaccination of IBH-affected horses.
However, major problems arose when producing and manufacturing the MAT molecules described in the prior art. In particular standard methods used in developing a downstream process (DSP) for manufacturing of the MAT molecules under good manufacturing practice (GMP) could not be applied. It was not possible to purify a homogeneous molecular species of the MAT molecules, evidently due to their anomalous physicochemical properties.
Several methods of purification could not be applied (see Example 5 herein) with the MAT molecules described in the prior art although different separation principles (e.g., size exclusion chromatography, RP-HPLC) were tested. Methods applied for determination of purity for recombinant proteins in general include chromatographic separation, e.g., RP-HPLC and electrophoretic separation (e.g. capillary zone electrophoresis, isoelectric focusing, SDS-PAGE under reducing or non-reducing conditions). Also, these analytical methods could not be applied on MAT molecules without molecule-specific adaptations. For the assessment of purity, an adapted specific SDS-PAGE test procedure had to be developed. This test procedure includes sample preparation with reducing agent and lithium dodecyl sulfate (LDS) and heating up to 75° C., resulting in multiple, reproducible sharp bands after electrophoretic separation. Staining with Coomassie blue dye leads to linear quantitative behavior (densitometry) in gels. Using a monoclonal antibody that allows for detection of the allergen module in a MAT molecule exhibited a main band and several minor bands. All bands migrate reproducibly to the same position as in the original gel also after re-loading a second gel with excised bands of the first gel. Surprisingly, in all of these bands with apparent lower and higher molecular weight, the full length MAT molecules were identified by excision of bands out of the gel, their tryptic digestion and subsequent analysis by mass spectrometry (nanoLC/ESI-MS-MS). From these experiments an untypical, anomalous behavior of different folding variants of MAT molecules in the SDS-PAGE can be concluded (“gel shifting”). Furthermore, in all batches of MAT molecules multimeric forms of the protein could be detected which were difficult to separate from monomeric forms.
For e.g. economic aspects, but also for regulatory requirements, it is necessary to improve (i) the manufacturing process of the MAT molecules and (ii) their suitability for standard analytical methods of purity determination. Additionally, for adapting the MAT molecules to specific target species, such as equines, adaption of the immunological targeting within the MAT technology is required.
Additionally, the MAT molecules are readily employed in allergies elicited by a known major allergen (e.g., cat dander allergy in humans by Fel d1). However, it seems difficult to employ the MAT molecules of the prior art in clinical settings, such as allergies, where for instance a variety of non-cross-reactive allergens are known to be involved, but the importance of such allergens in eliciting the allergy is unknown (i.e. the major allergens are unknown).
The objective underlying the invention is to provide improved MAT molecules useful as active agents in pharmaceutical composition, such as vaccines, which overcome the problems of the prior art.